The role of viruses in carcinogenesis has long been accepted because of the direct mechanistic effects of often a single viral gene in cell transformation. The involvement of bacteria in carcinogenesis, however, remains controversial partly because there is no clear agreement on the molecular mechanism(s) by which they might promote the development of cancer. Nonetheless, epidemiological evidence has linked prior and chronic bacterial infection to tumor formation. Although most attention has been focused on H. pylori, other bacterial infections have been shown to cause cancer in animal models. Bacterial toxins are known to modulate intracellular signaling pathways directly in a way that could promote tumor development. However, their carcinogenic role is not fully studied. Pasteurella multocida toxin (PMT) is an intracellular acting bacterial protein known for its potent mitogenic properties in vitro and in vivo and its ability to induce strong anchorage-independent growth for certain type of cells. These properties suggest that PMT might have the potential to act as a tumor promoter especially in the case of chronic infections. The detailed mechanism behind mitogenic properties of PMT is unknown. Recent reports show that PMT exerts its biological effects, in part, via the deamidation of a conserved glutamine residue in the subunit of heterotrimeic G proteins, including Gq, Gi, G12, and G13, and leads to a constitutively active phenotype of the G proteins. We have previously shown that rPMT induces protein and ATP synthesis, cell migration and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces a sustained phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (S6). This phosphorylation is inhibited by rapamycin and Torin1, two specific inhibitors of mammalian target of rapamycin (mTOR). The PMT-mediated mTOR activation was observed in MEF WT but not in MEF Gq/11 knockout cells, consistent with our results indicating that PMT-induced mTOR activation proceeds via the deamidation of Gq/11 and leads to the activation of PLC to generate diacylglycerol (DAG) and inositol trisphosphate (IP3), two known activators of PKC pathway. An increasing body of evidence supports the idea that extracellular matrix (ECM) proteins are major players in the global control of intercellular communication and integration of environmental signals. It was not known whether rPMT-treated cells are able to express and secrete into the medium a substrate(s) capable of activating autocrine and/or paracrine signaling. We have found that the conditioned medium from rPMT-treated cells activates mTOR and MAPK signaling, but not membrane-associated tyrosine kinase signaling. Surprisingly, this diffusible factor(s) can activate mTOR and MAPK pathways even in MEF Gq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in 3T3 cells, along with other genes involved in cell proliferation and cancer biology. The elevation of CTGF, an ECM protein upregulated in certain cancers and in fibrosis, was confirmed by RT-PCR and Western blot analysis. In accord with rPMT-induced mTOR activation, upregulation of CTGF was mediated by deamidation of Gq/11, and was independent of TGF, a well-known inducer of CTGF. Collectively these results prompt us to study a possible involvement of PMT in the differentiation of fibroblast to myofibroblast. Myofibroblasts possess ultrastructural features intermediate between fibroblasts and smooth muscle cells and have been defined by their ability to express contractile proteins, particularly -smooth muscle actin(-SMA) 1 protein. This contractile phenotype is functionally important for the closure of cutaneous wounds. In addition, these cells represent an activated fibroblast phenotype with high synthetic capacity for extracellular matrix (ECM) proteins, growth factors/cytokines, growth factor receptors, integrins. In cultured 3T3 cells, PMT induces a time-dependent steady increase in -SMA protein and mRNA expression. The up-regulation of -SMA was dose-dependent and remained elevated for up to 5 days without repeat dosing, consistent with a stably differentiated state. This effect of PMT was associated with morphological changes of cellular hypertrophy and well-formed actin stress fibers, characteristic of myofibroblasts. In addition to 1 and 2 SMA, PMT also upregulates gamma 2 SMA (enteric) mRNA expression. It has been reported that myofibroblast differentiation also requires the presence of Fibronectin Extra Domain A. Western blot analysis and RT-PCR show that PMT treatment did not induce fibronectin or fibronectin EDA expression. It is worth mentioning that PMT-induced myofibroblast differentiation is independent of TGF 1 expression as we previously described. We believe that PMT cause myofibroblast differentiation by a mechanism different from that of TGF 1 and may involve heterotrimeric G protein deamidation mediated by PMT. To study the role of Gq/11 in PMT-induced myofibroblast differentiation, we used WT and Gq/11 knockout MEF cells. We observed that PMT induces a substantial increase in SMA in MEF cells in comparison to the untreated cells. MEF knockout cells without any treatment express a low level of SMA. Following PMT treatment, SMA levels increase in knockout cell but remains low in comparison to the WT MEF cells exposed to the same concentration of PMT. This result suggests that SMA expression in Knock out cells might be due the PMT-induced deamidation of other heterotrimeric G proteins. Myofibroblasts play a central role in repair of wound tissues through their capacity to produce strong contractile forces and recruit cell migration. Thus, we examined contractile activity of myofibroblast using collagen gel contraction. Indeed, PMT stimulated contraction of the collagen gel to 60+/-12.1% of the initial area 48 hours after PMT stimulation. This result supports our previous observation showing that PMT treatment increase ATP levels. The ATP produced after PMT treatment will certainly sustain myofibroblast contractibility since this process is energy dependent. We next sought to determine the role of MPT-induced mTOR activation on myofibroblast differentiation. The effect of rapamycin on myofibroblast differentiation was investigated by treating starved 3T3 cells with PMT in the presence or absence of rapamycin, a well-known mTOR inhibitor. Rapamycin pretreatment did not have any effect on SMA protein and mRNA expression following PMT treatment. Its worth mentioning that we did not observe any effect of rapamycin on PMT-induced ECM proteins including CTGF expression protein and mRNA. However, rapamycin did inhibit PMT-induced intracellular proteins including survivin and Glut1. This result is in agreement with our previous results showing that mTOR is partially involved in protein expression and cell proliferation. The myofibroblast, long known for having a role in wound-healing, and for its presence in fibrotic conditions and tumor stroma, is becoming a focus for translational research, not least through its increasingly documented role as a tumor-promoting cell. While the involvement of the myofibroblast in these pathological processes is pushing the myofibroblast into the limelight of translational medicine as a target for potential anti-fibrotic and anti-cancer therapy, there are still numerous indications from the literature that the myofibroblast is a poorly understood cell in terms of its differentiation.